Egorin M J, Snyder S W
Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
Cancer Res. 1990 Jul 1;50(13):4044-9.
N,N',N''-Triethylenethiophosphoramide ([14C]thiotepa) accumulation by L1210 cells is a biphasic process. A very rapid initial phase is followed by a much slower second phase that reflects accumulation of radioactivity in a form that is not lost or exchanged when cells are resuspended and incubated in drug-free medium for up to 8 h. In this study we attempted to characterize this nonexchangeable radioactivity. Nuclei (10(7)) isolated from L1210 cells and incubated with [14C]thiotepa did not accumulate 14C during incubations of up to 5 h. Similarly, nuclei isolated from 10(7) L1210 cells that had been shown to accumulate nonexchangeable 14C after incubation with [14C]thiotepa did not show an increase in nuclear-associated 14C. Eighty to 85% of nonexchangeable 14C in L1210 cells incubated with [14C]thiotepa was soluble in ethanol or chloroform:methanol (2:1, v/v), and although most of this cell-associated nonexchangeable 14C was precipitated by trichloroacetic acid, subsequent treatment of that precipitate with methanol solubilized most of the 14C so that only 15 to 20% remained with the final precipitate. When chloroform:methanol-soluble nonexchangeable 14C was analyzed with thin-layer chromatography systems suitable for thiotepa or simple lipids, all radioactivity remained at the origin. In contrast, when analyzed with one- and two-dimensional thin-layer chromatographic systems suitable for complex lipids, all chloroform:methanol-soluble radioactivity was associated with a single lipid spot. This lipid cochromatographed with phosphatidylethanolamine, reacted with ninhydrin but not with 4-(p-nitrobenzyl)pyridine or the Dragendorff choline reagent, and was digested by phospholipases C and D, all of which lead to its identification as phosphatidylethanolamine. This extensive labeling of phosphatidylethanolamine in L1210 cells incubated with [14C]thiotepa can be explained by liberation of [14C]aziridine from [14C]thiotepa, hydrolysis of the [14C]aziridine to [14C]ethanolamine, and incorporation of that radiolabeled material into phosphatidylethanolamine via the normal cellular synthetic pathways for that lipid. This information implies that thiotepa serves, at least in part, as a prodrug for aziridine and has implications as to the mechanism of thiotepa-induced cytotoxicity in that aziridine is a monofunctional alkylating agent incapable of producing interstrand, intrastrand, or protein-DNA cross-links.
L1210细胞对N,N',N''-三亚乙基硫代磷酰胺（[14C]噻替派）的摄取是一个双相过程。最初是非常快速的阶段，随后是一个慢得多的第二阶段，该阶段反映了放射性以一种在细胞重悬于无药培养基中并孵育长达8小时时不会丢失或交换的形式积累。在本研究中，我们试图表征这种不可交换的放射性。从L1210细胞中分离出的细胞核（10(7)个）与[14C]噻替派一起孵育，在长达5小时的孵育过程中没有积累14C。同样，从10(7)个L1210细胞中分离出的细胞核，这些细胞在与[14C]噻替派孵育后已被证明积累了不可交换的14C，但未显示核相关14C增加。与[14C]噻替派一起孵育的L1210细胞中80%至85%的不可交换14C可溶于乙醇或氯仿：甲醇（2:1，v/v），尽管这种与细胞相关的不可交换14C大部分被三氯乙酸沉淀，但随后用甲醇处理该沉淀物可使大部分14C溶解，因此最终沉淀物中仅保留15%至20%。当用适用于噻替派或简单脂质的薄层色谱系统分析氯仿：甲醇可溶的不可交换14C时，所有放射性都留在原点。相反，当用适用于复合脂质的一维和二维薄层色谱系统分析时，所有氯仿：甲醇可溶的放射性都与单个脂质斑点相关。这种脂质与磷脂酰乙醇胺共色谱，与茚三酮反应，但不与4-(对硝基苄基)吡啶或德拉根多夫胆碱试剂反应，并被磷脂酶C和D消化，所有这些都导致其被鉴定为磷脂酰乙醇胺。在与[14C]噻替派一起孵育的L1210细胞中磷脂酰乙醇胺的这种广泛标记可以通过从[14C]噻替派中释放[14C]氮丙啶、将[14C]氮丙啶水解为[14C]乙醇胺以及通过该脂质的正常细胞合成途径将该放射性标记物质掺入磷脂酰乙醇胺来解释。该信息表明噻替派至少部分地作为氮丙啶的前药，并且对噻替派诱导的细胞毒性机制具有启示意义，因为氮丙啶是一种单功能烷基化剂，不能产生链间、链内或蛋白质-DNA交联。
